The aim of this proposal is to develop an effective method to overproduce recombinant human hemoglobin in Escherichia coli. The protein then can be harvested in quantities sufficient to use as therapeutic hemoglobin, which could serve as a temporary blood substitute. Hemoglobin expressed in E. coli is degraded unless sufficient heme, which can be synthesized by the cell, is available for incorporation into the apoprotein. The approach is to increase the heme in the cell by moving a heme transport system from another organism into E. coli that contains the hemoglobin genes, and supplying heme in the media. This should result in additional stable hemoglobin being produced. A portion of the work reported in this proposal centers around sperm whale myoglobin, which is simpler but structurally similar to hemoglobin, and thus serves as a model for hemoglobin function. Work was conducted on E. coli transformed with two plasmids, one containing either the myoglobin gene or the hemoglobin genes, and one containing heme transport genes from the intestinal pathogen, Plesiomonas shigelloides. Data obtained using a spectrophotometric assay indicated that substantial increases in globin production occurred when the strains were grown in media containing heme versus in media with no added heme. The increases ranged from 40% to 170%, depending on the strain being tested. These data suggest that the strains were incorporating the transported heme into the apoglobin protein(s), allowing larger quantities of stable hemoglobin to be produced. The specific aims of this proposal are as follows: 1) Use western blotting to examine levels of soluble hemoglobin protein in E. coli containing the recombinant plasmids encoding the hemoglobin genes and the P. shigelloides heme transport system; 2) Identify strains of E. coli that are the most appropriate for overexpressing hemoglobin. 3) Identify the most promising bacterial heme transport system in terms of allowing efficient transport of heme and high levels of stable hemoglobin. 4) Modify promoters of the heme transport genes to allow greater expression under high iron conditions. 5) Create a single plasmid that contains both the heme transport genes and the hemoglobin genes, and/or create a strain of E. coli that contains the heme transport genes from the most promising heme transport system incorporated into the E. coli chromosome. The last specific aim addresses technical problems encountered during the project.